PROJECT SUMMARY The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. One aspect of this proposal is to clarify how proteins are translocated post-translationally from the cytosol across the bacterial plasma membrane or the eukaryotic endoplasmic reticulum (ER). We have determined X-ray structures of the protein-conducting SecY channel, with and without the ATPase SecA and substrate, and used biochemical experiments to study bacterial and eukaryotic translocation, providing the basis for the present proposal. In eukaryotes, there is a retro-translocation pathway, called ERAD (for ER associated degradation). We have recapitulated ERAD with purified components and have determined a structure of the Hrd1 ubiquitin ligase, which likely forms a protein-conducting channel. Here, we will address central questions of these protein translocation pathways: Specific aim #1: What is the mechanism of post-translational translocation? Based on crystal structures and biochemical experiments, we have proposed a ?push-and-slide? model by which SecA mediates post-translational translocation in bacteria. We will perform single-molecule FRET experiments to test this model and clarify how SecA couples its ATPase and mechanical cycles. To address the mechanism of post-translational translocation in eukaryotes, we will determine cryo-EM structures of the yeast Sec complex in the absence and presence of bound substrate and perform biochemical experiments. Specific aim #2: How are proteins moved through the membrane during ERAD? We will test whether Hrd1 forms a protein-conducting channel by determining cryo-EM structures of Hrd1 in complex with its partners. We will use reconstituted systems with purified proteins to understand how substrates are selected for ERAD, use crosslinking methods to determine the path of a polypeptide through the retro-translocon, and test the postulated role of auto-ubiquitination of Hrd1 in channel gating. Specific aim #3: How are ERAD substrates moved from the ER membrane to the proteasome? We will determine cryo-EM structures of the Cdc48 complex together with substrate, analyze how substrate processing is initiated, how the ATPase complex extracts polypeptides from the ER membrane, and how polypeptides are transferred to the proteasome. We will investigate the role of binding partners of Cdc48 and of shuttling factors in the transfer of substrates to the 26S proteasome, and test whether some substrates can be transferred directly to the 20S proteasome. The mechanism of protein translocation is of great medical importance. Many diseases, including cystic fibrosis, are caused by the misfolding of ER proteins and their degradation. The pathway is also hijacked by certain viruses and toxins, and a better understanding may lead to new drugs allowing interference.